Soft tissues have complex mechanical properties that depend largely on their underlying structural organization. However, the relationship between microscale (collagen) properties and macroscale (tissue) behavior remains unclear. A multiscale modeling approach, which relates these two scales directly, was recently applied successfully to evaluate biaxial tensile testing of cell-seeded collagen Type I gels (tissue- equivalents, or TEs). TEs are particularly useful model systems whose compositional and organizational properties can be easily modified. Unfortunately, these tissue analogs have not been used to fully evaluate more complex loading protocols such as those experienced by many tissues in vivo, including compression and shear forces. One example is the supraspinatus tendon (SST) of the rotator cuff, which has a high rate of degeneration and injury that may be due to the complex loading environment of the shoulder. As a bridge to evaluating the SST (and other soft tissues that function in multiaxial loading environments), the use of TEs will allow for the development, testing, and analysis of progressively more complex systems. For example, a recent study that added agarose during collagen gel formation reported an increase in tissue stiffness with minimal changes to collagen fiber architecture. This presents a valuable modification to the TE model system, by enabling us to investigate the mechanical and structural contribution of non-collagenous extracellular matrix (ECM) (e.g., proteoglycans, glycosaminoglycan, etc.) to tissue properties. Therefore, the objective of this study is to characterize the three-dimensional behavior of soft tissues under complex loads by quantifying the mechanical and structural properties of organizationally- and compositionally-varying tissue-equivalents (TEs) using experimental analysis and multiscale modeling. We hypothesize that differences in the microscale collagen fiber organization and the relative stiffness of the non-collagenous ECM components (represented by agarose) will result in large changes in the macroscale mechanical and localized structural response of TEs in complex loading (i.e., indentation and shear). The following aims are proposed: Specific Aim #1: Fabricate tissue-equivalents (TEs) of varying organizational structure (collagen fiber alignment) and composition (collagen-only or collagen-agarose), and quantify their three-dimensional structural and mechanical properties under complex loading conditions (i.e., indentation and shear). Specific Aim #2: Utilize multiscale computational models of three-dimensional TE behavior to characterize the relationships among structural, compositional and mechanical properties. This study will provide valuable insight into the nature of, and relationships between, the mechanical, structural and compositional properties of TEs, which will lay the necessary groundwork for future experimental and computational evaluations of similar properties in healthy and injured native tissues. Such studies will provide data that will greatly aid clinicians and scientists in strategies for treating, repairing, or replacing soft tissues. PUBLIC HEALTH RELEVANCE: Degeneration and injury of soft tissues commonly result in pain and disability, and the inability to effectively prevent or treat these injuries is likely due to an incomplete understanding of tissue properties on multiple length-scales. The purpose of this study is to use mechanical/structural analyses and computer modeling to evaluate the microscale and macroscale properties of bioengineered tissue analogs, which present a simplified model system for native tissues. This study will provide valuable insight into the nature of, and relationships between, the mechanical, structural and compositional properties of tissue-equivalents, which will lay the necessary groundwork for future evaluations of similar properties in healthy and injured native tissues. Such studies will provide data that will greatly aid clinicians and scientists in strategies for treating, repairing, or replacing soft tissues.